Method for ex-vivo expansion of regulatory t cells with enhanced suppressive function for clinical application in immune mediated diseases

ABSTRACT

The invention provides methods for the ex-vivo expansion of CD4+CD25+ Tregs. The invention provides a method for producing ex vivo expanded Tregs that may be used to inhibit unwanted human immune responses against self-antigens or allergens. Additionally, the ex vivo expanded Tregs may provide treatment for inflammatory/automimmune diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61,179,165, filed May 28, 2009.

FIELD OF THE INVENTION

The invention relates to methods for enhancing the suppressive functionof regulatory T cells. In particular, the invention provides a methodfor ex-vivo expansion of regulatory T cells, which cells demonstrateenhanced suppressive characteristics.

BACKGROUND OF THE INVENTION

Regulatory T cells, or Tregs, are known to be critical in maintaining atolerance to self-antigens by suppressing the activation of the immunesystem. In person with inflammatory/autoimmune diseases, such assystemic lupus erythematous, multiple sclerosis, rheumatoid arthritis,asthma, ulcerative colitis, and Crohn's Disease, there is a functionaldefect or frequency decrease in the transcription factor Foxp3expressing CD4+CD25+ Tregs. In such autoimmune diseases, the person'simmune system fails to recognize cells, or parts of cells as theperson's own resulting in tissue destruction.

The therapeutic effects of administration of CD4+CD25+ Tregs has beenshown in the treatment of a variety of animal disease models including,without limitation, rheumatoid arthtritis, asthma, and graft-versus-hostdisease (“GVHD”). Use of Tregs in treatment applications is problematicbecause they are present as only a very small percentage, approximately1 to 2%, of human peripheral blood mononuclear cells. Thus, methods ofactivating and expanding, or proliferating, Tregs ex-vivo have beendeveloped for use in the treatment of certain diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the frequency of CD4+Foxp3 +Tregs from thepopulation of Example 1.

FIG. 2 is a graph showing the purity CD4+ Tregs of Example 2.

FIG. 3 is a graph showing the Treg expansion of Example 3.

FIG. 4 is a graph showing the percentage of ex-vivo expanded cells ofExample 3 that expressed Foxp3.

FIG. 5 is a graph showing the inhibitory activity of the expanded Tregsof Example 4.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The invention provides methods for the ex-vivo expansion of CD4+CD25+Tregs, compositions resulting from the method and methods for use of thecompositions. The cells resulting from use of the method demonstrateenhanced suppressive activities compared to fresh Treg cells. Theinvention provides a method for producing ex vivo expanded Tregs thatmay be used to inhibit unwanted human immune responses againstself-antigens or allergens. Additionally, the ex vivo expanded Tregs mayprovide treatment for inflammatory/automimmune diseases.

In one embodiment, the invention provides a methods comprising,consisting essentially of, and consisting of: a) obtaining a populationof T cells from an individual; b) isolating and purifying from the Tcell population a subpopulation of CD4+CD25+ Tregs; and c) expanding theTreg cells of the subpopulation, wherein the expanded Treg cells exhibitenhanced suppressive activity compared to a population of freshlypurified, unexpanded Tregs from the individual.

It is a discovery of the invention that Tregs taken from a person withautoimmune/inflammatory diseases including, without limitation SystemicLupus Erythematous (“SLE”), Multiple Sclerosis (“MS”), asthma,Rheumatoid Arthritis (“RA”), Crohn's Disease (“CD”), and UlcerativeColitis (“UC), display enhanced suppressive activities to inhibit T cellproliferation when compared to freshly purified Tregs from the sameperson. It is a further discovery of the invention that the Tregs can beexpanded 100 to 1,000 fold ex-vivo while having enhanced suppressivefunction.

In a first step of the method of the invention, a population of T cellsis obtained. The T cells may be obtained from any suitable sourceincluding, without limitation, an individual's peripheral blood, thymus,lymph nodes, spleen or bone marrow. Preferably, the T cells are obtainedfrom the peripheral blood of a person. More preferably, the T cells areobtained from an individual with an autoimmune/inflammatory diseases andmost preferably from an individual with one or more of SLE, MS, asthma,RA, CD and UC whether the disease is in remission or during a period inwhich the disease is active. Most preferably, human CD4+CD25+ T regs arepurified from whole blood units or leukpaks.

A subpopulation of CD4+CD25+ Tregs is then isolated from the T cellpopulation using any convenient separation techniques based on Tregspecific cell markers, including, without limitation, flow cytometry byany convenient method. For example, any number of kits for carrying outsuch isolation and purification are commercially available. Thecommercially available kits include, without limitation, Miltenyi Tregkit with Auotmacs, ClinMACS, and the like. Preferably, the isolation andpurification is carried out until a population that is greater than 40%positive for Foxp3 and greater than 90% positive for CD4 is obtained.

The Treg subpopulation is then expanded ex-vivo using an autoantigenspecific regulatory T cell stimulatory composition. Preferably, thecomposition antigen-specifically binds and activates the T cell receptorcomplex. More preferably, the expansion is carried out in the presenceof effective amounts of a first and a second activator and aco-stimulator activator. By effective amount is meant an amounteffective to stimulate the regulatory T cells to the degree desired.

The first activator is a TCR/CD3 activator that may be a multivalentantibody or ligand for TCR/CD3 including, without limitation, antigennon-specific activators such as an anti-CD antibody, andantigen-specific activators, such as an MHC-peptide multimer in whichthe peptide is an autoimmune/inflammatory disease associated peptide.Preferably, the TCR/CD3 activator is an anti-CD3 antibody.

The composition includes a second activator that is an additionalsuitable regulatory T cell stimulator. This component may include,without limitation an interleukin. Preferably IL-2 is used. The IL-2typically is used in recombinant form and used in an amount of about1,000 IU/ml.

A multivalent antibody or ligand specific for a TCR co-stimulator may beused as the co-stimulator activator. Preferably, the co-stimulatoractivator is CD28. The activator may be enhanced by use of one or moreadditives including, without limitation, rapamycin, a P13 kinaseinhibitor, anit-IL6 and the like.

The TCR/CD3 activator and TCR co-stimulator activator preferably areimmobilized on a three-dimensional solid surface, more preferably on abead. Suitable beads are commercially available such as cell ExpanderDynalbeads (Invitrogen). The Treg to bead ratio is preferably is 1:3.Optimal bead size will depend on a consideration of the size required toefficiently congregate the antibodies and typically is about 1 to 10microns in diameter. The anti-CD3 and anti-CD28 antibody ratio on thesurface of the bead may vary from about 1 to 20 or about 20 to 1.

The expansion is carried out to at least a 100 fold expansion,preferably to a greater than 1,000. The expansion will depend upon thestimulation and length of the culture. However, other variations may beintroduced into the culture the longer the length of the culture and,thus, a culture time that permits obtaining the most cells without asubstantial introduction of other unfavorable factors is preferred.

The invention will be clarified by consideration of the following,non-limiting examples.

EXAMPLES Example 1

Approximately 5 to 10 peripheral blood samples of 50 cc each werecollected from 9 persons with SLE, 7 persons with RA, 7 persons withasthma, 10 persons with MS, and 7 persons with CD as well as from 11persons with no autoimmune disease.

CD4+ Tregs were isolated and purified from the peripheral bloodmononuclear cells (“PBMCs”) of the samples. The PBMCs were isolated fromblood samples by density gradient centrifiguation with Ficoll Hypaque(Amersham). The CD4+CD25+ Tregs were purified from PBMCs by autoMACSusing the human CD4+CD25+ regulatory T cell isolation kit (MiltenyiBiotec, Auburn, Calif.) according to the manufacturer's instructions.Briefly, CD4+ T cells were first negatively isolated from PBMCs bydepleting non CD4 cells with the mixture of monoclonal antibodiesagainst human CD8, CD14, CD16, CD19, CD36, CD56, CD123, TCRγ/δ andCD235a. Human CD4+CD25+ Tregs were then positively isolated withanti-human CD25 antibody-conjugated microbeads from the enriched CD4+ Tcell population. The frequency of Foxp3-expressing CD4+ Tregs wasassessed using intracellular Foxp3 staining and analyzed by flowcytometry. The results are displayed in FIG. 1 below in which thepercentage of CD4+Foxp3+ Tregs in the CD4+ pool population from eachperson is shown.

Example 2

Approximately 5 to 10 peripheral blood samples of 50 cc each werecollected from 9 persons with SLE, 7 persons with RA, 7 persons withasthma, 8 persons with MS, and 7 persons with CD as well as from 25persons with no autoimmune disease.

CD4+ Tregs were purified from the PBMCs of the samples. Purification wascarried out using as set forth in Example 1. The purity of purified CD4+Tregs at day 0 was assessed using intracellular Foxp3 staining andanalyzed by flow cytometry and the results are displayed on the graph inFIG. 2. Approximately 40 to 75% of the purified cells expressed Foxp3.

Example 3

Approximately 5 to 10 peripheral blood samples of 50 cc each werecollected from persons with SLE, RA, asthma, MS, CD, and UC. CD4+ Tregswere purified from the PBMCs of the samples. Purification was carriedout using as set forth in Example 1.

The CD4+Foxp3+ Tregs were cultured in X-VIVO 15™ media (CambrexBio-Whitaker, East Rutherford, N.J.) supplemented with 10% pooled humanAB serum (Cambrex) in the presence of 1000 IU/Ml of human rIL-2(Proleukin). In addition, anti-CD3/anti-CD28 coated beads (Dynal, Oslo,Norway) were added at a 1:3 cell to bead ratio. After 3 weeks of cultureat 37° C. in an incubator, the expanded Tregs reached over 100 to 1,000fold expansion, or 1 billion cells, as shown in the graphs in FIG. 3. InFIG. 4 is a graph depicting that an average of 40 to 50% of the ex-vivoexpanded cells expressed Foxp3 by intracellular staining at 2 weeks.

Example 4

The Tregs of Example 3 were evaluated at week 2 with standardizedsuppressive assays as follows. For anti-CD3 stimulated cultures,allogenic CD4+CD25+ human T cells (5×10⁴ cells/well) from the same donorwere used as responder cells, 1 mg/ml anti-CD3 (OKT3), and allogenichuman dendritic cells (from one donor, 5×10³ cells/well) and seriallydiluted expanded Tregs were put in 96-well plates in triplicate. Foralloantigen stimulated cultures, CD4+CD25+ human Treg cells (5×10⁴cells/well) from one donor, 5×10³ human dendritic cells from anotherdonor, and serially diluted expanded Tregs were plated in triplicate in96-well plates.

The results, shown in the graphs of FIG. 5, demonstrate that the ex vivoexpanded human Tregs had potent inhibitory activity to inhibit T cellproliferation in in vitro functional assays. Additionally, the Tregs'inhibitory activity was enhanced as compared to that of freshly purifiedTregs from the same persons. This suppressive activity may be used toinhibit unwanted human immune responses in autoimmune/inflammatorydiseases and provide a clinical therapeutic potential forautoimmune/inflammatory diseases.

1. A method comprising: a) obtaining a population of T cells from anindividual with an immune-mediated disease selected from Systemic LupusErythematous, Multiple Sclerosis, asthma, Rheumatoid Arthritis, Crohn'sDisease, or Ulcerative Colitis; b) isolating and purifying from the Tcell population a subpopulation of CD4+CD25+ Tregs; and c) expanding theTreg cells of the subpopulation, wherein the expanded Treg cells exhibitenhanced suppressive activity compared to a population of freshlypurified, unexpanded Tregs from the individual.
 2. The method of claim1, wherein the expansion is between about 100 to 1,000 fold.
 3. Themethod of claim 1, wherein the expansion was carried out for a period ofabout 3 weeks.
 4. A method of treating an individual withimmune-mediated disease, the method comprising: a) obtaining apopulation of T cells from an individual with an immune-mediated diseaseselected from Systemic Lupus Erythematous, Multiple Sclerosis, asthma,Rheumatoid Arthritis, Crohn's Disease, or Ulcerative Colitis; b)isolating and purifying from the T cell population a subpopulation ofCD4+CD25+ Tregs; c) expanding the Treg cells of the subpopulation; andd) administering a portion of the CD4+CD25+ regulatory T cells to ahuman being treated for the immune-mediated disease.
 5. The method ofclaim 4, wherein the immune-mediated disease is Systemic LupusErythematous.
 6. The method of claim 4, wherein the immune-mediateddisease is Multiple Sclerosis.
 7. The method of claim 4, wherein theimmune-mediated disease is asthma, Rheumatoid Arthritis.
 8. The methodof claim 4, wherein the immune-mediated disease is Rheumatoid Arthritis.9. The method of claim 4, wherein the immune-mediated disease is Crohn'sDisease.
 10. The method of claim 4, wherein the immune-mediated diseaseis Ulcerative Colitis.